matrix metalloproteinase (stromelysin-1) increases the albumin permeability of isolated rat...

Matrix metalloproteinase (stromelysin-1)increases the albumin permeability of isolated rat glomeruli

RAM SHARMA, KO SUZUKI, HIDEAKI NAGASE, and VIRGINIA J. SAVIN

MILWAUKEE, WISCONSIN, and KANSAS CITY, KANSAS

Matrix metalloproteinases (MMPs) secreted by connective tissue cells are capable of acting on extracellular matrix components of glomerular basement membrane at a slow rate and thus may play a role in the control of protein permeability and in the progression of certain kinds of glomerulonephritis. We have used an in vitro assay to measure the direct effect of three MMPs and human neutrophil elastase on glomerular albumin permeability (Palbumin)" Glomeruli were isolated from normal male Sprague-Dawley rats and suspended in isolation medium with or without interstitial collagenase, gelatinase-A, stromelysin-1, or elastase and were incubated at 37 ° C for up to 4 hours. A tissue-specific inhibitor of matrix metalloproteinases (TIMP-I) and a plasma proteinase inhibitor, ~2-macroglobulin (~2M), were used to block the activity of MMPs. Pa~bumin was calculated from the change in glomerular volume in response to an applied oncotic gradient. In this study stromelysin-1 (10 l~g/ml) and elastase (5 l~g/ml) increased Pa~bum~. significantly. Strome- lysin- I increased Patbum~. after 4 hours, whereas elastase had an effect after 2 hours. Lower concentrations of stromelysin-I or shorter incubation time had no effect on Pa~b~mJ.. Incubation for up to 4 hours with interstitial collagenase (10 ~g/ml) or gelatinase-A (10 l~g/ml) had no effect on Pa~b~m~.. Coincubation with TIMP-I and oL2M blocked the stromelysin-l-mediated increase in Palbumin" We conclude that stromelysin-1 is capable of affecting the glomerular filtration barrier directly and that it may play an important role in causing proteinuria in glomerular diseases. (J Lab Clin Med 1996; 128:297-303)

Abbreviations: c~2M = e2-macroglobulin; APMA = 4-amino phenyl mercuric acetate; BSA = bovine serum albumin; GBM = glomerular basement membrane; MMP = matrix metalloproteinase; MMP-1 = interstitial collagenase; MMP-2 = gelatinase-A; MMP-3 = stromelysin-1; Pa~bum~n = glomerular albumin permeability; ~a~bum~n = albumin reflection coefficient; TIMP-I = tissue inhibitor of metalloproteinases; z~V = glomerular volume change

From the Department of Medicine, Division of Nephrology, Medical College of Wisconsin, Milwaukee; and the Departments Biochemistry and Molecular Biology, University of Kansas Med- ical Center, Kansas City. Supported by Public Health Service Grant 22040, by grants from the Kansas Affiliate of the American Heart Association, and by the Biomedical Research Support Grant Program at the Univer- sity of Kansas Medical Center. Submitted for publication Nov. 16, 1995; revision submitted March 19, 1996; accepted April 1, 1996. Reprint requests: Ram Sharma, Phi), Division of Nephrology, De- partment of Medicine, CVRC Building, Room 466 C, Medical College of Wisconsin, 8701 Water Town Plank Road, Milwaukee, WI 53226. Copyright © 1996 by Mosby-Year Book, Inc. 0022-2143/96 $5.00 + 0 5/1/74240

M atrix metalloproteinases make up a group of related endopeptidases that have the ability to degrade extracellular matrix

components. They are secreted from the connective tissue cells (fibroblasts) as an inactive zymogen I and can be activated by organomercurial compounds such as A P M A or proteinases. The activation of zymogen to active enzyme results in a loss of 10 to 12 kd of molecular mass. The activity of MMPs can be inhibited by a specific TIMP-1 produced by the same cells 2

The well-characterized metalloproteinases are interstitial collagenase (MMP-1), gelatinase-A (MMP-2), and stromelysin-1 (MMP-3). Collagenase

297

298 Sharma et al,

specifically digests interstitial collagen types I, II, and III. 3 More recently it has also been shown to digest collagen types VII and X. 4'5 Gelatinase-A preferen- tially degrades gelatin but is capable of degrading na- tive collagen types IV, V, VII, and XI and to a lesser extent proteoglycans, laminin, and elastin. 6"1° Strome- lysin-1 is capable of degrading many components of the GBM including collagen types IV, IX, and X laminin, proteoglycans, and fibronectin. 11"16

The GBM is composed primarily of collagen type IV, with lesser amounts of collagen type V, laminin, fibronectin, proteoglycans, and heparan sulfate. ~7 The integrity of the GBM, as well as the integrity of the endothelial and epithelial cells of the glomerular capillary, appears to be essential to the normal filtration barrier. The GBM contributes to both the size and charge barriers that restrict the passage of plasma proteins into the urinary space, is

The synthesis of various neutral metalloproteinases has been described in rat kidney glomeruli 19 as well as in cultured human glomerular mesangial cells. 2° The role of gelatinase-A 21 and stromelysin-114'22 in the degradation of bovine GBM has been studied. These enzymes are slow reacting and require at least 16 to 18 hours to generate measurable GBM degradation products. It has been postulated that degradation of GBM by various MMPs may contribute to the pathogenesis of glomerulonephritis. ~8

There is no direct evidence in the literature that the transient increase in various MMPs can directly affect the glomerular filtration barrier and lead to an increase in the permeability of plasma proteins before the measurable degradation of GBM, which requires elevated levels of these enzymes for a longer period of time. Using an in vitro method, we studied the short-term effect of three purified MMPs and elastase on the glomerular filtration barrier by measuring the increase in glomerular capil-

lary Palbumin-

METHODS

Experimental animals. Normal male Sprague-Dawley rats (180 to 250 gm body weight) were given free access to Purina chow (Purina Mills, St. Louis, Mo.) and water. At the time of study, animals were weighed and anesthetized with metofane (Pitman-Moore Inc., Mundelein, ILL). Kid- neys were removed via abdominal incision.

Isolation of glomeruli. After the kidney capsule was removed, the outer 1 to 2 mm of renal cortex was excised and cut into fine fragments. These fragments were passed through consecutive stainless steel screens of 80, 120, and 200 mesh size as described in previous studies. 19,2° The glomeruli were recovered from the top of a 200 mesh screen. The isolation of glomeruli was carried out at room temperature in medium that contained, in millimoles per

J Lab Clin Med September 1996

liter, sodium chloride, 115; potassium chloride, 5; sodium acetate, 10; dibasic sodium phosphate, 1.2; sodium bicar- bonate, 25; magnesium sulfate, 1.2; calcium chloride 1; glucose, 5.5; L-alanine, 6; sodium citrate, 1; sodium lac- tate, 4; and BSA, 4 gm/dl. The pH of the medium was adjusted to 7.4 by bubbling with 5% CO2/95% O2. The oncotic pressure was measured with a membrane colloid osmometer (model 4100; Wescor Inc., Logan, Utah) and expressed in millimeters of mercury.

Incubation medium. The incubation medium was identical to the isolation medium except for the addition of the agents to be tested. The proenzymes proMMP-1, proMMP-2, and proMMP-3 were purified to homogeneity from culture medium of human rheumatoid synovial fibroblasts stimulated with rabbit macrophage-conditioned medium as described previously. 8'26'z7

Purified proMMP-1 was fully activated by treatment with plasmin and stromelysin-1 at 37 ° C for 6 hours. 27 Plasmin was inactivated by 2 mmol/L diisopropylfluorophosphate; stromelysin-1 was removed by using an anti-human stromelysin-1 affinity column. Collagenase was further purified by Sephacyl S-200 gel filtration equilibrated with 50 mmol/L Tris-HCl buffer (pH 7.5), 0.4 NaC1, 10 mmol/L Ca 2+, 0.05% 23 lauryl ether, and 0.02% NaN 3.

ProMMP-2 was activated by adding 1 mmol/L APMA (Sigma Chemical Co., St. Louis, Mo.) just before use. ProMMP-3 was activated by 1 mmol/L APMA at 37 ° C for 24 hours, 28 and APMA was removed by gel filtration with a Sephacyl S-200 column as described for MMP-1.

Human neutrophil elastase was purified by the method of Baugh and Travis z9 and was a gift from Dr. G. Salvesen of Duke University. TIMP-1 was purified from culture medium of human fibrosarcoma cell line HT-1080 as described previously. 3° Human oL2M was purified as described earlier 31'32 and was given to us by Dr. Jan J. Enghild of Duke University.

Purified MMPs and elastase used in this study were tested for their enzymatic activity and were found to retain their activity in incubation medium containing 4 gm/dl BSA. In previous studies we have shown that the enzymatic activity of MMPs is abolished by TIMP-133 and aaM. 34

Measurement of AV. Incubated glomeruli with or without various test agents in 4% BSA were transferred to an observation chamber and were allowed to adhere to a glass coverslip coated with poly-L-lysine (1 mg/ml). Indi- vidual glomeruli were observed with videomicroscopy in 4% BSA and then again 2 to 3 minutes after the medium was replaced with medium containing 1 gm/dl BSA. This exchange of medium from 4% to 1% BSA produced an oncotic gradient (A~r) across the glomerular capillary wall and resulted in an influx of fluid and an increase in glomerular capillary volume. The volume of each glomerulus before and after the exchange of medium was calculated from the average mean diameter (D) of the video image by using the formula V = 3/4 7r (D/2) 3. The AV (increase) in each glomerulus in response to an oncotic gradient (A~r) for each experimental condition was calculated as follows:

J Lab Clin Med Volume 128, Number 3 Sharma et al. 299

E

.<

0.8

0.6

0.4

0.2

0.0 Control 2.5 5.0 10

S t r o m e l y s i n - 1 (ug/ml)

Fig. L Effects of increasing concentrations of stromelysin-1 on Palburnin- Bars indicate mean -+ SEM of Palbumin" *Statistically different from control, p < 0.01.

A V = [(Wfina I - Winitial)/Winitial] X 1 0 0 % .

Measurement of O'albumi n and Palbumln" The rationale and calculations for these measurements have been de- tailed in an earlier report, as Because the increase in AV is proportional to the oncotic gradient (A~r) across the capillary wall, we used this principle to calculate ~ralb,mi . by dividing the volume of experimental glomeruli by the volume change in control glomeruli in response to an identical oncotic gradient, as follows:

O'albumi n = AVexper imenta l /mVcont ro 1 Pa]bumin was calculated from (ralb,ml n and was defined as Palbumin = (1 -- O'albumin)

When the reflection coefficient, cralb,min, is zero, Palbumln is 1.0. When (ra~b,mi . is 1.0, albumin cannot cross the membrane and Palbumin is zeroY

Statistics. Average P~b,mm was calculated from four to five glomeruli from at least 3 or more rats in each experimental condition, and values were compared by using anal- ysis of variance. All values are expressed as mean + SEM, and a value of p < 0.01 among groups was considered significant.

RESULTS

Effect of various concentrations of stromelysin-1 on Panbumin" Glomerul i were incubated for 4 hours at 37 ° C with different concentra t ions (2.5, 5, or 10 ixg/ml) of stromelysin-1, and Palbumin was calculated. As shown in Fig. 1, only the highest con-

centrat ion of stromelysin-1 (10 Ixg/ml) caused a significant increase in Palbumin (0.61 _+ 0.09, N = 15) as compared with results in controls (0.03 +_ 0.03, N = 15).

Effect of three different MMPs on Palbumin. Glomeru l i were incubated with or without 10 Ixg/ml collagenase, gelatinase-A, or stromelysin-1 for 4 hours at 37 ° C, and Palbumin was calculated. As shown in Fig. 2, only stromelysin-1 caused a significant increase in Palbumin (0.62 ----- 0.06, N = 15) as compared with results in control glomeruli (0.03 _ 0.035, N = 15), whereas interstitial collagenase (0.1 __- 0.11, N = 15) and gelatinase-A (0.12 ___ 0.06, N = 15) had no significant effect on Palbumin"

Minimum time required by stromelysin-I to cause its effect on Palbumin" Glomeruli were incubated with or without stromelysin-1 (10 p~g/ml) for 1, 3, or 4 hours at 37 ° C before calculation of Palbumin" As shown in Fig. 3, there was a slight increase in Palbumin after 3 hours (0.13 _ 0.08, N = 15) as compared with results in controls (0.03 ___ 0.035, N = 14) but the significant increase in Palbumin was evident only after 4 hours of incubation (0.65 --- 0.07, N = 32).

Inhibition of stromelysin-l-mediated increase in Pallournln by TIMP-1 and ot2M. Glomeruli were incubated with or without stromelysin-1 for 4 hours at 37 ° C. Addi- tional glomeruli were added to preincubated stromelysin-1 with either TIMP-1 (12.5 ~g/ml) for 30 minutes or with a2M (200 ~g/ml) for 1 hour at

300 Sharma et al, J Lab Clin Med

September 1996

o ~

"i

m

1° 1 0.8

0.6

0.4

0.2

0.0 Control MMP-1 MMP-2 MMP-3

MMPs at 10ug/ml

Fig. 2. Effect of collagenase (MMP-1), gelatinase-A (MMP-2), and stromelysin-1 (MMP-3) on Palbumin" Bars indicate mean - SEM of P,lbumin" *Statistically different from control, p < 0.01.

°u

°u

E

, , 0

1.0-

0.8

0.6

0.4

0.2

0.0 Control 1 hr 2 hr 4 hr

S t r o m e l y s i n - 1 (10ug/ml)

Fig. 3. Minimum time required by 10 ixg/ml of stromelysin-1 to cause an increase in P~bumin. Bars indicate mean + SEM of PaZbumin" *Statistically different from control, p < 0.01.

37°C and fu r the r incuba ted for 4 hours at 3 7 ° C be fo re m e a s u r e m e n t of Palbumin" AS shown in Fig. 4, s t romelysin-1 inc reased Palbumin signif icantly (0.66 --+ 0.09, N = 15 vs a cont ro l va lue of 0.03 + 0.35, N =

15), and this effect of s t romelysin-1 was inh ib i ted by bo th T IMP-1 (0.09 _ 0.04, N = 14) and e~2M (0.1 -+ 0.08, N = 15). N e i t he r T I M P - 1 nor et2M by them- selves had any effect on Palbumin"

J Lab Clin Med Volume 128, Number 3 Sharma et al. 301

1.0-

° ~

,g}

0.8

0.6

0 . 4 '

0.2'41 ~ **

0.0 Control MMP MMP+TIMP MMP+LMG

S t r o m e l y s i n - l ( 1 0 u g / m l )

Fig. 4. Inhibition of stromelysin-l-mediated increase in Palbumin by TIMP-1 and ct2M. Bars indicate mean +_ SEM of Palbumin" MMP, Stromelysin-1. *Statistically different from control, p < 0.01. **Signifi- cantly different from *, p < 0.01.

Effect of elastase on Palbum~n" Because infusion of elastase and cathepsin G induces proteinuria in rats 19'2°'36 we studied the effect of elastase on the glomerular albumin permeability in vitro. Glomeruli were incubated with 2.5, 5.0, or 10.0 i~g/ml elastase for 2 hours at 37 ° C, and Palbumin was calculated. As shown in Fig. 5, elastase at a 5 ixg/ml concentration caused a significant increase in Palbumin (0.73 --+ 0.09, N = 15) as compared with the control value (0.00 + 0.06, N = 15).

DISCUSSION

Proteinuria is a nonspecific manifestation of glomerular disease that results from an increase in the permeability of the glomerular capillary to albumin and other plasma proteins. The role of the serine proteinases (elastase and cathepsin G) released by intrinsic glomerular cells 19'2° or by infiltrating neutrophils 36 in damage to the glomerular filtration barrier, resulting in proteinuria in vivo, has been documentedJ 9'a°'36 In this study we have shown that purified elastase from human neutrophils directly affects the glomerular filtration barrier without the participation by secondary hemodynamic or immu- nologic factors, causing an increase in the Palbumin value in isolated rat glomeruli.

MMPs are neutral proteinases capable of degrading components of extracellular matrix including the GBM. The presence of GBM-degrading neutral

proteinase was first documented by Lovett et al. in the rat, 2° in human mesangial cells,37and then in normal rat glomeruli. 19 The presence of neutral proteinases in the urine of nephrotic rats suggests a possible role of these enzymes in proteinuria. 23 GBM-degrading neutral proteinase has been purified and characterized from rat mesangial cells. 21 The role of neutral proteinases in the degradation of the GBM has been well documented. 14'22 Because degradation of the GBM leads to proteinuria and destructive glomerular diseases, a study of the role of MMPs in Palbumin s eem s appropriate.

Interstitial collagenase and gelatinase-A have been purified and characterized from the kidney and glomeruli and appear to be the most plentiful of the metalloproteinases. The presence of levels of mRNA for stromelysin-1 in normal rat glomeruli has been reported by Jones et al. 38'39 The level of mRNA encoding stromelysin-1 in control and in diabetic (streptozocin-injected) rat glomeruli has also been reported. 4° The degradation of type IV collagen by gelatinase-A and by stromelysin-1 has been documented, and their potential role in glomerulonephritis has been suggested, 14'22 but there is no evidence that stromelysin-1 or any other MMP can alter Palbumin" In this study interstitial collagenase and gelatinase-A had no effect, whereas stromelysin-1 caused a significant increase in P~lbumt~. Increased protein permeability was evident

302 Sharma et al. J Lab Clin Med

September 1996

° ~

° I A~

t~

I i

a ~

2

1.0

0.8

0.6

0 , 4 '

0.2

0.0 Control 2.5 5.0 1 0

Human Neutrophil Elastase (ug/ml)

Fig. 5. Effect of human neutrophil elastase on Palbumin" Bars indicate mean -+ SEM of Palbumin" *Statis- tically different from control, p < 0.01.

within 4 hours of incubation, whereas degradation of the GBM required at least 16 to 18 hours of incubation a4 with stromelysin-1.

The lack of effect of interstitial collagenase and gelatinase-A on P~lbumi~ Can be explained by the fol- lowing: (1) collagenase has its effect only on collagen (types I, II, III, VII, X) and no effect on collagen type IV, a major component of the GBM; (2) gelatinase-A has its major effect on collagen (types IV, V, VII, X) and elastin and a limited effect on proteoglycans; (3) gelatinase-A degrades collagen type IV only at a specific site; (4) stromelysin-1, however, degrades collagen (types IV, IX, X) and all of the other components of the GBM including laminin, proteoglycans, fibronectin, and heparan sulfate; (5) in addition, stromelysin-1 cleaves type IV collagen at multiple sites 3s and thus may be more potent in its effect on the alteration of the glomerular permeability barrier. Alternatively, stromelysin-1 may act primarily through degradation of noncollagen components of the GBM such as laminin and proteoglycans, which are not the substrates for interstitial collagenase and gelatinase-A but may play an important role in the structural integrity of the filtration barrier by maintaining cell-to-cell and cell-to- GBM interaction.

TIMP-1 is secreted from the mesangial cells, which are known to secrete MMPs. 36 Inhibition of the effect of stromelysin-1 on Palbumin by TIMP-1 would suggest that stromelysin-1 along with TIMP-1 plays a significant role in the catabolism of GBM. In

this study, the increase in Palbumi. with stromelysin-1 and its inhibition by TIMP-1 indicates the impor- tance of matrix components in maintaining the permeability barrier. The regular turnover of the GBM and mesangial matrix is a slow process and is con- trolled by basal levels of secretion of MMPs and their specific inhibitor, TIMP-1. Transient aug- mented release of MMPs during glomerular inflam- mation may result in their enhanced activity on various components of the GBM and may lead to proteinuria; if release of MMPs exceeds the level of TIMP-1, the accelerated activity of these enzymes may lead to destructive glomerular diseases in vivo.

We postulate that the stromelysin-l-mediated increase in Palb~,i~ is caused by the effect of stromelysin-1 on the components of the GBM (such as adhesion molecules or integrins), which are important in cell-to-cell and cell-to-GBM interaction. This action of stromelysin-1 may result in altered adhesion charac- teristics, slit pore junctions, or receptors of glomerular epithelial cells before the degradation of the GBM.

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